Research Paper

Mediators of Inflammation 3, 125-129 (1994)

To elucidate the modulatory role of histamine-degradingenzymes in airway constrictor responses, human bron-chial strips were studied under isometric conditions invitro. Pretreatment of tissues with the histamine N-me-thyltransferase (HMT) inhibitor SKF 91488 specificallypotentiated the contractile responses to histamine, caus-ing a leftward displacement of the concentration-response curves, whereas the diamine oxidase inhibitoraminoguanidine had no effect. This potentiation was at-tenuated by mechanical removal of the epithelium. TheHMT activity was detected in the human bronchi, whichwas less in the epithelium-denuded tissues than the epi-thelium-intact tissues. These results suggest that HMTlocalized to the airway epithelium may play a protectiverole against histamine-mediated bronchoconstriction inhumans.

Key words: Asthma, Bronchial smooth muscle contraction,Histamine N-methyltransferase

Histamine N-methyltransferasemodulates human bronchialsmooth muscle contraction

J. Tamaoki,cA A. Chiyotani, E. Tagaya,K. Isono and K. Konno

First Department of Medicine, Tokyo Women’sMedical College, Tokyo 162, Japan

ca Corresponding Author

Introduction

Histamine is released from mast cells and basophilsby a variety of stimuli including antigen cross-linkedIgE, and induces airway smooth muscle contraction,microvascular leakage and mucus production." Thushistamine probably plays a principal role in thepathogenesis of immediate asthmatic responses. Inaddition, because increased bronchial responsive-ness to histamine is a well-characterized feature ofasthma, bronchial provocation by histamine has rou-tinely been used to facilitate its diagnosis.

It has been known that histamine can be metabo-lized by two major pathways in the body;4’5 50 to 70%of histamine is metabolized by histamine N-methyltransferase (HMT, EC 2.1.1.8), located in thesmall intestine, liver, kidney and leukocytes, into N-methylhistamine, and the remaining 30 to 45% ismetabolized by diamine oxidase (DAO, EC 1.4.3.6),also called histaminase, located in intestinal mucosa,placenta, liver, skin, kidney, neutrophils andeosinophils, to imidazole acetic acid. Recent studieson guinea-pig trachea showed that HMT but notDAO decreases bronchoconstrictor responses to his-tamine and antigen challenge.6,: However, it remainsunknown which enzyme is responsible for the de-gradation of histamine in the human airway andwhere the enzyme is located. Therefore, humanbronchial strips were studied under isometric condi-tions in vitro to elucidate the modulatory role ofhistamine-degrading enzymes in the histamine-medi-ated bronchoconstriction.

Materials and Methods

Preparation of tissues: Human lung tissues wereobtained from 23 patients at thoracotomies per-

formed because of carcinoma. After surgical removal,pieces of macroscopically normal lung tissues wererapidly immersed in Krebs-Henseleit solution con-sisting of the following composition (in mM): NaCl,118; KC1, 5.9; MgSO4, 1.2; CaC1,., 2.5; NaH,.PO, 1.2;NaHCO3, 25.5; and D-glucose, 5.6; gassed with amixture of 95% O-5% CO,. at 37C. Cartilaginousbronchi, 2 to 4 mm in internal diameter, were thendissected free of parenchyma, fat and surroundingconnective tissues, and cut helically at a 45 pitchto obtain bronchial strips measuring 2 to 3 mm inwidth and approximately 20 mm in length. Betweentwo and eight strips were dissected from each speci-men and mounted in 14 ml organ chambers contain-ing Krebs-Henseleit solution aerated, with 95%02-5% CO2 at 37C (pH 7.4, Pco,. 38 Torr, PO > 500Torr). Contractile responses were continuouslymeasured isometrically with a force-displacementtransducer (Nihon Kohden, JB-652T, Tokyo, Japan)and were recorded on a pen recorder (NihonKohden, WT-685G). The tissues were allowed toequilibrate for 60 min while they were washed withKrebs-Henseleit solution every 15 min, and the rest-ing tension was adjusted to 1 g. A contractile re-sponse was determined as the difference betweenpeak tension developed and resting tension. Allexperiments were conducted in the presence ofindomethacin (3 x 10-6M) and ranitidine (6 x 10-5 M)to avoid prostaglandin release and histaminetachyphylaxis,8 respectively.

Effects of histamine-degrading enzyme inbibitors:Following the equilibration period, histamine wasadded to the chamber in a cumulative manner atconcentrations ranging from 10-s to 10-3 M in half-molar increments at 5 min intervals or 2 min after

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J. Tamaoki et al.

stable plateau was achieved, whichever was thelonger period. After establishing the firstconcentration-response curves, tissues were washedwith Krebs-Henseleit solution until the tensionreturned to the baseline level and, 60 min later,SKF 91488 (10-4 M), an inhibitor of HMT,9’1or aminoguanidine (10-4 M), an inhibitor of DAO,11was added. Twenty min later, the secondconcentration-response curves for histamine weregenerated in a similar manner. In a control experi-ment, two successive concentration-response curvesfor histamine were likewise generated without addi-tion of an inhibitor.To test whether the inhibition of HMT activity also

alters the contractile responses to other spasmogenicagonists, the effect of SKF 91488 (10-4 M) on theconcentration-response curves for acetylcholine andKC1 were examined, with the same time sequence forthe contractile responses to histamine.At the end of these experiments, each bronchial

strip was blotted on a gauze pad and weighed. Activetensions were normalized for tissue weight and ex-pressed as grams tension per gram of tissue weight.To characterize the concentration-response curves,the maximal contractile response (Emax) and the nega-tive logarithm of molar concentration of agonist re-quired to produce 50% of Emax (pD,) were determinedby linear regression analysis.To assess concentration-dependent effects of his-

tamine-degrading enzyme inhibitors, contractile re-sponses of bronchial strips to 10-5 M histamine weredetermined in the absence and presence of eitherSKF 91488 or aminoguanidine (10- to 10-3 M). In thisexperiment, after determining the first responses tohistamine, tissues were washed, applied with a singleconcentration of either inhibitor and, 20 min later,the second responses to histamine were determined.To assess whether the modulation of the contrac-

tile responses to histamine by SKF 91488 was asso-ciated with the epithelium, concentration-responsecurves for histamine were constructed in the absenceand presence of SKF 91488 (10.4 M) in tissues withthe epithelial cells denuded. Bronchial strips weregently rubbed off their luminal surface with a moistcotton swab, and confirmation of the successfulremoval of the epithelium was histologically per-formed in randomly selected tissues.

Measurement ofHMT activity: The activity of HMTwas measured in bronchial tissues with and withoutepithelium according to the method by Fukuda etal.’ Briefly, excised bronchi were homogenized in aglass homogenizer, with four volumes of ice-coldphosphate buffered saline (0.05 M, pH 7.4) contain-ing 1 mM dithiothreitol and 1% polyethyleneglycol.The homogenate was centrifuged at 4C (120 000 xg,1 h), and the supernatant was dialysed three times for8 h against 100 volumes of the buffer. The reaction

of HMT was carried out at 37C in 0.5 ml of a mixtureof 0.1 ml of the supernatant, 0.3 ml of 0.1 M phos-phate buffered saline containing 0.1 mM pargylineand 0.1 mM aminoguanidine (pH 7.4), 0.05 ml of1 mM histamine and 0.05 ml of S-adenosyl-t-methionine. After incubation, N*-methylhistaminewas separated from histamine by high-performanceliquid chromatography on a weak cation exchanger(Toyo-Soda Co., TSKgel CM2SW, Tokyo), with37.5 mM citric acid, 1.25% imidazole and 20%acetonitrile, with the mobile phase at a flow rate of1.0 ml/min. The fluorescence intensity of the reactionmixture was then measured using a post-columnderivatization with o-phthalaldehyde and 2-mercaptoethanol, and the HMT activity was ex-pressed as pmol of N-methylhistamine formed per hper mg of protein as determined by the method ofLowry et al.

Drugs: The following drugs were used: histaminediphosphate, indomethacin, acetylcholine chloride,KCl, aminoguanidine (Sigma Chemical Co., St Louis,MO); and ranitidine (Glaxo Co., Tokyo). SKF 91488(S-[4[(N,N-dimethylamino)-butyl] isothiourea) was agift from Smith Kline Co. (Philadelphia, PA).

Statistics: All values were expressed as means + S.E.Comparative statistical analysis was performed usingANOVA followed by either Tukey’s test for multiplecomparisons or by Student’s t-test; n refers to thenumber of preparations, andp < 0.05 was consideredstatistically significant.

Results

Muscle contraction: As demonstrated in Fig. 1, inthe control experiment both the Emax and the pD,values of the second histamine concentration-response curves of human bronchial strips were notsignificantly different from those of the firstconcentration-response curves. Thus, the histamine-induced tachyphylaxis was not observed in the pres-ence of indomethacin and ranitidine. Addition of SKF91488 (10-4 M) and aminoguanidine (10-4 M) did notalter the resting tension. Pretreatment of tissues withSKF 91488 potentiated the contractile responses tohistamine, causing a leftward displacement of thehistamine concentration-response curves with thepD,. values being increased from 5.0 +_ 0.1 to 5.8 +_ 0.3(p

HMT and airway smooth muscle

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0

Control

SKF 91488

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Log [Histamine] (M)

FIG. 1. Effects of SKF 91488 (10 M) and aminoguanidine (10 M) oncontractile responses of human bronchial strips to histamine. After estab-lishing baseline responses to histamine (open circles), tissues werewashed, either inhibitor was added and, 20 min later, the secondconcentration-response curves were generated (closed circles). Tissueswithout addition of an inhibitor served as control experiments. Values areexpressed as percentage of the maximal baseline response. Each pointrepresents mean + S.E.; n= 10 for control and SKF 91488, and n= 9 foraminoguanidine.

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- 100ExE 80o

140O

120

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SKF 91488 *** ***

Aminoguanidine

-8 -7 -6 -5 -4 -3

Log [Inhibitor] (M)

FIG. 2. Concentration dependent effects of SKF 91488 and aminoguanidineon contractile responses to 10 M histamine. Values are expressed aspercent of the baseline responses obtained before the addition of aninhibitor. Data are means+S.E.; n=8 for each column. *p

J. Tamaoki et al.

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80

60

40

0

-8 -7 -6 -5 -4 -3

Log [Histamine] (M)

FIG. 3. Effect of SKF 91488 (10 M) on contractile responses to histaminein bronchial strips with the epithelium removed. After establishing baselineresponses to histamine (open circles), SKF 91488 was added and thesecond concentration-response curves were generated (closed circles).Values are expressed as percent of the maximal baseline response. Eachpoint represents mean + S.E.; n 11.

1200

000

800

600

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2OO

Epithelium intact Epithelium denuded

FIG. 4. Histamine N-methyltransferase (HMT) activity in human bronchialtissues. Activity of the enzyme was measured by high-performance liquidchromatography based on post-column derivatization with o-phthalaldehyde. Data are means + S.E.; n 11 for epithelium-intact tissuesand n 12 for epithelium-denuded tissues. *p < 0.05, significantly differentfrom values for epithelium-intact tissues.

of the concentration-response curves was small com-pared with the epithelium-intact tissues; SKF 91488potentiated histamine-induced contraction by 6.3-fold in epithelium-intact strips and by 3.2-fold inepithelium-denuded tissues.

Tissue HMT activity: The activity of HMT in humanbronchi determined by high-performance liquidchromatography was 920 _+ 230 pmol/mg protein/h(n 11) for the epithelium-intact tissues and 280 + 70pmol/mg protein/h (n 12) for the tissues withoutepithelium. There was a significant difference be-tween these values (p< 0.05, Fig. 4).

DiscussionThe present in vitro studies demonstrate that HMT

probably localized to human airway epithelium mayplay a protective role in the histamine-mediatedbronchoconstriction through the degradation of his-tamine to its inactive metabolite. This notion is basedon the following findings. First, pretreatment ofbronchial strips with the HMT inhibitor SKF 914889,1potentiated the histamine-induced contraction in aconcentration dependent fashion without affectingcontractile responses to other spasmogenic agonistsincluding acetylcholine and KCl; secondly, the mag-nitude of the SKF-induced leftward displacement ofhistamine concentration-response curves was sig-nificantly less in bronchial strips with the epitheliummechanically removed than in the epithelium-intacttissues; and thirdly, the activity of HMT was meas-ured by high-performance liquid chromatographyusing post-column derivatization with o-phthalaldehyde and it was found that the HMT activ-ity of human bronchial tissues was greatly decreasedby removal of the epithelium.

It has been known that histamine is metabolizedby two major enzymes, HMT and DAO, located ona variety of mammalian tissues and inflammatorycells. TM HMT catalyses methyl transfer from S-adenosyl-t-methionine to histamine to form N-methylhistamine, which is further metabolized bymonoamine oxidase to N-methylimidazole aceticacid,15 and DAO also metabolizes histamine to N-methylimidazole acetic acid. 16 In the present study, itwas found that, in contrast to the effect of SKF 91488,aminoguanidine at concentrations sufficient to in-hibit DAO activity11 had little effect on the contractileresponses to histamine. Therefore, histamine may bemetabolized principally through the HMT pathway inhuman airways, as is also true in the brain. 17

The airway epithelium has been shown to inhibitbronchoconstrictor responses to a variety of stimuliby releasing epithelium-derived relaxing factor,which is not a product of arachidonic acid and is notnitric oxide,18-2 and by metabolizing tachykininswith neutral endopeptidase.2 Concerning the hista-mine metabolism, there are conflicting reports onguinea-pig tracheal epithelium. Lindstr6m et al.showed the aminoguanidine-induced potentiation ofthe contractile responses to histamine, but Ohrui etal. recently reported the lack of aminoguanidine’seffect and showed the presence of HMT activity andits mRNA in the epithelium by in situ hybridization,and Sekizawa et al. reported a similar finding inantigen-induced bronchoconstriction in vivo. Thepresent findings on human bronchi were in agree-ment with the latter two reports. Although DAOactivity was not measured, the results of the musclebath experiments suggest that the epithelial HMTactivity is more important than DAO in limiting the

128 Mediators of Inflammation Vol 3. 1994

HMT and airway smooth muscle

biological actions of histamine in human airways. Intissues without epithelium, the, HMT activity was stillpresent and the SKF 91488-induced potentiation ofthe contractile responses to histamine was small butstill significant. These findings suggest that HMTlocalized to other cell types such as endothelial cellscould also contribute to histamine degradation.

In conclusion, the histamine-degrading enzymeHMT in the airway epithelium may play a modulatoryrole in the bronchoconstriction in human airwaysthrough a metabolism of histamine. The authorstherefore speculate that airway hyperresponsivenessto histamine in asthmatic subjects could be due, atleast in part, to the epithelial damage-associated lossof HMT activity.

References1. Braude S, Royston D, Coe C, Barnes PJ. Histamine increases lung permeability by

Ha-receptor mechanism. Lancet 1984; 2: 372-374.2. Shelhamer JH, Marom Z, Kaliner M. Immunologic and neuropharmacologic stimu-

lation of glycoprotein release from human airways in vitro. J Clin Invest1980; 66: 1400-1408.

3, Scadding JG. Definition and clinical categories of asthma. In: Clark TJH, GodfreyS, eds. Asthma. London: Chapman Hall, 1983; 1-11.

4. Beavens MA. Histamine: its role in physiologic and pathologic process. MonogrAllergy 1978; 13: 1-20.

5. Zeinger RS, Yurdin DL, Colten HR. Histamine metabolism. II. Cellular andsubcellular localization of the catabolic enzymes, histaminase and histaminemethyltransferase in human leukocytes. JAllergy Clin Immuno11976; 58: 172-179.

6. Ohrui T, Yamauchi K, Sekizawa K, et al. Histamine N-methyltransferase controlsthe contractile responses of guinea pig trachea to histamine. JPharmacol Exp Ther1992; 261: 1268-1272.

7. Sekizawa K, Nakazawa H, Ohrui T, et aL Histamine N-methyltransferase modulateshistamine- and antigen-induced bronchoconstriction in guinea pigs in vivo. Am RevResptrDis 1993; 147: 92-96.

8. Knight DA, Stewart GA, Thompson PJ. Histamine tachyphylaxis in human airwaysmooth muscle: the role of Ha-receptors and the bronchial epithelium. Am RevRespir Dis 1992; 146: 137-140.

9. Beaven MA, Shaff RE. New inhibitors of histamine N-methyltransferase. BiochemPharmacol 1979; 28: 183-188.

10. Beaven MA, Shaff RE. Inhibition of histamine methylation in vivo by the Dimapritanalog, SKF compound 91488. Agents Actions 1979; 9: 455-460.

11. Schuler W. Zur Hemmung der Deaminooxydase (Histaminase). Experientia 1952;$: 230-232.

12. Fukuda H, Yamatodani A, Imamura I. High-performance liquid chromatographicdetermination of histamine N-methyltransferase activity. J Chromatogr 1991; 56"7:459-464.

13. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with theFolin phenol reagent. JBiol Chem 1951; 193: 265-275.

14. White MV, Slater JE, Kaliner MA. Histamine and asthma. Am Rev Respir Dis 1987;135: 1165-1176.

15. Schayer RW, Karjala SA. Ring N methylation: major route of histamine metabo-lism. JBiol Chem 1956; 221: 307-313.

16. Douglas WW. Histamine and 5-hydroxytryptamine (serotonin) and their antago-nists. In: Gilman AG, Goodman LS, Rail TW, Murad F, eds. The PharmacologicalBasis of Therapeutics. New York: Macmillan Publishing Co., 1985; 605-638.

17. Schwartz JC. Histaminergic mechanisms in brain. Ann RevPharmacol Toxico11977;17: 325-339.

18. Flavahan NA, Aarhus LL, Rimele TJ, Vanhoutte PM. Respiratory epithelium inhibitsbronchial smooth muscle tone. JAppl Physiol 1985; 58: 834-838..

19. Shikano K, Ohlstein EH, Berkowitz BA. Differential activity of epithelium-derivedrelaxing factor and nitric oxide in smooth muscle. BrJ Pharmacol 1987; 92:483-485.

20. Munakata M, Masaki Y, Sakuma I, et al. Pharmacological differentiation of epithe-lium-derived relaxing factor from nitric oxide. JAppl Physiol 1990; 69: 665-670.

21. Sekizawa K, Tamaoki J, Graf PD, Basbaum CB, Borson DB, Nadel JA.Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction inferret trachea. JPharmacol Exp Ther 1987; 243: 1211-1217.

22. Lindstr6m EG, Andersson RGG, Granrus G, Grundstr6m N. Is the airway epithe-lium responsible for histamine metabolism in the trachea of guinea pig? AgentsActions 1991; 33: 170-172.

ACKNOWLEDGEMENTS: The authors thank Smith Kline Co. for providing with SKF91488. This work supported in part by grant No. 04670476 from the Ministry ofEducation, Science and Culture, Japan.

Received 4 October 1993;accepted in revised form 20 December 1993

Mediators of Inflammation. Vo13. 1994 129

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